Synthesis, purification and testing of point-modified messenger RNA
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Abstract
Ribonucleic acids (RNA) are involved in a huge variety of important cellular processes. They have been a subject of scientific research for decades now, and the huge variety of non-canonical nucleosides, namely modifications, that have been discovered, add another layer to the regulatory capabilities of the various RNA species. However, there are gaps in the knowledge regarding some aspects of point-modified messenger RNAs (mRNAs), as mechanistic studies have been performed mainly with short mRNA surrogates. This PhD thesis aims to develop a suitable synthesis method of point-modified mRNA to investigate the influence of modifications on translation efficiency.
The mRNA of the enhanced green fluorescent protein with coupled internal ribosomal entry site (IRES-eGFP) served as a model and was first divided retrosynthetically into several fragments, with the middle fragment intended to introduce a modification into the mRNA with point accuracy. Subsequently, the mRNA was reconstituted in a 3 way one pot splint ligation and purified via the newly developed method of real time gel elution. The splint ligation approach was adapted from shorter RNA and adjusted to long RNA species. The purification method was investigated with respect to its practicability and safety against RNA damage, with particular emphasis on gel particles on the one hand and oxidative damage due to irradiation during elution on the other. The obtained point-modified mRNAs were analyzed with focus on the number and position of the respective modification. After confirmed correct incorporation of the desired modification and reconstitution of the full-length mRNA, the obtained modified mRNAs were translated in vitro and the protein yield was measured against the protein yield of an unmodified reference mRNA. In particular, 2'-O-methylations introduced into the Kozak sequence in and around the start codon revealed remarkable results for cap-independent IRES-driven translation, which had not been reported before.